Volume 23, Issue 10, Pages 1653-1662 (October 2015) Ubiquitin-like Molecule ISG15 Acts as an Immune Adjuvant to Enhance Antigen-specific CD8 T-cell Tumor Immunity  Daniel O Villarreal, Megan C Wise, Rebekah J Siefert, Jian Yan, Laurence M Wood, David B Weiner  Molecular Therapy  Volume 23, Issue 10, Pages 1653-1662 (October 2015) DOI: 10.1038/mt.2015.120 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Generation and expression of ISG15 encoding DNA vaccine plasmids. (a) Schematic illustration of ISG15 protein and the amino acid sequences of wild-type ISG15 (wtISG15) and mutated ISG15 (mutISG15). The IgE leader sequences are underlined. The C-terminal ubiquitin-like conjugation site is bold and underlined. The mutation sites introduced into the conjugation motif for mutISG15 (unconjugated form) are in red. (b) Map of ISG15 constructs. (c) Expression of ISG15 constructs examined by western blot analysis. The lowest band represents free ISG15. (d) Detection of secreted wtISG15 and mutISG15 from transfected RD cells were confirmed via enzyme-linked immunosorbent assay. Data represent the means with standard error of the mean for two replicate assays. RD, human rhabdomyosarcoma. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Codelivery of ISG15 DNA vaccination promoted E7-specific CD8 T-cell immune responses secreting IFNγ production. (a) Immunization schedule for DNA vaccine adjuvant study. C57BL/6 mice (n = 4–5/group) were immunized twice at 2-week intervals with HPV16 construct with or without wtISG15 or mutISG15 adjuvant constructs via IM/EP delivery. One week after last vaccination, spleens were harvested to analyze the Ag-specific CD8 T-cell responses. (b) The frequency of E7-specific IFNγ (spot forming cells/106 splenocytes) responses induced after each vaccination was determined by IFNγ ELISpot assay in response to E7 pooled peptide containing the specific CD8 HPV16 E7 epitope (RAHYNIVTF). Data represent two independent experiments with four to five mice per group. *P < 0.05; **P < 0.01. Error bars indicate standard error of the mean. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 ISG15 induces polyfunctional HPV16 E7-specific CD8 T-cells. (a) Schematic diagram of gating strategy used to identify Ag-specific CD8 T-cell populations. (b–d) Column graphs show the percentages of HPV16 E7-specific CD8 T-cells releasing total cytokines IFNγ (b), TNFα (c), and IL-2 (d) after stimulation with DbE749-57-specific peptide. (e) Column chart show polyfunctional subpopulations of single-, double-, or triple-positive CD8 T-cells releasing effector cytokines: IFNγ, TNFα, and IL-2 to E749-57-specific stimulation. Pie charts represent proportion of each cytokine population. Experiments were performed at least two times with similar results with four to five mice per group. All cell counts are relative and not absolute. *P < 0.05 compared with HPV16 group. Error bars indicate standard error of the mean. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 ISG15 induces HPV16 E7-specific CD8 T-cells undergoing cytotoxic degranulation following immunization. E7-specific CD8 T-cell responses measured by intracellular cytokine and CD107a staining after stimulation of splenocytes with DbE749-57 restricted (CD8) peptide were examined in all groups of animals 1 week after final immunization. (a) Ag-specific cytolytic degranulation of CD8 T-cells measured by staining for degranulation marker expression, CD107a. (b and c) Column graph shows the frequency of cytolytic CD8 T-cells simultaneously expressing only IFNγ (b) or the frequency of polyfunctional cytokine producing and/or CD107a expressing CD8 T-cells (c). Experiments were performed at least twice with similar results with four to five mice per group. All cell counts are relative and not absolute. *P < 0.05; **P < 0.01 compared with HPV16 group. Error bars indicate standard error of the mean. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 ISG15 augments the formation of the E7-specific effector CD8 T-cell population. Groups B6 mice (n = 4–5) were immunized twice with HPV16, HPV16/wtISG15 or HPV16/mutISG15 at two-week intervals. One week after last immunization, both splenocytes and peripheral blood mononuclear cells were strained for CD8, DbE749-57 tetramer, and the effector KLRG1 marker. (a) Representative flow plot showing H2-Db-RAHYNIVTF-restrticted HPV16 E7-specific CD8 T-cells in the spleen 1 week after final immunization, or (b) in data represented as a scatter plot graph. (c,d) Representative dot plots (c) and compiled data of the percentages of E7 tetramer-specific KLRG1+CD8+ effector phenotype population in the spleen (d). (e,f) The percentages of total DbE749-57 tetramer-binding CD8 T-cells from the peripheral blood (e) and tetramer-specific effector CD8 T-cells (f). Data are representative of at least two experiments. All cell counts are relative and not absolute. *P < 0.05; **P < 0.01. Error bars indicate standard error of the mean. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 The therapeutic effects induced by ISG15 in tumor-bearing mice. (a) Schematic representation for therapeutic study. (b) Tumor growth measurement after therapeutic DNA/electroporation vaccination (n = 10). (c) Schematic representation for CD8 T-cell depletion with therapeutic vaccination. (d) Tumor growth curve of vaccinated groups (n = 5) without CD8 T-cells. (e and f) Schematic representation for T-cell adoptive transfer study (e). Approximately 4 × 106 CD8 T-cells from vaccinated mice were purified from splenocytes and adoptively transferred into tumor-bearing T-cell immunodeficient B6 Rag1 KO mice (n = 5) and assessed for tumor growth (f). All tumor-bearing mice were injected subcutaneously with 5 × 104 TC-1 cells. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate standard error of the mean. Molecular Therapy 2015 23, 1653-1662DOI: (10.1038/mt.2015.120) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions